Glucosyl stevia composition

ABSTRACT

Glucosyl  stevia  compositions are prepared from steviol glycosides of  Stevia rebaudiana  Bertoni. The glucosylation was performed by cyclodextrin glucanotransferase using the starch as source of glucose residues. The short-chain glucosyl  stevia  compositions were purified to &gt;95% content of total steviol glycosides. The compositions can be used as sweetness enhancers, flavor enhancers and sweeteners in foods, beverages, cosmetics and pharmaceuticals.

RELATED APPLICATIONS

This application is a continuation-in-part application of and claims thebenefit of priority from:

co-pending U.S. patent application Ser. No. 14/073,423, filed on Nov. 6,2013, which is a continuation application of U.S. patent applicationSer. No. 13/567,707, filed on Aug. 6, 2012 and issued as U.S. Pat. No.8,647,844 on Feb. 11, 2014, which is a divisional application of U.S.patent application Ser. No. 13/029,263, filed on Feb. 17, 2011 andissued as U.S. Pat. No. 8,257,948 on Sep. 4, 2012; and

co-pending U.S. patent application Ser. No. 13/589,754, filed on Aug.20, 2012, which is a continuation of U.S. patent application Ser. No.13/074,179, filed on Mar. 29, 2011 and issued as U.S. Pat. No. 8,318,459on Nov. 27, 2012, which is a continuation-in-part application of U.S.patent application Ser. No. 13/029,263, filed on Feb. 17, 2011 andissued as U.S. Pat. No. 8,257,948 on Sep. 4, 2012.

The contents of each of these applications are incorporated herein byreference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a process for producing a highly purified foodingredient from the extract of the Stevia rebaudiana Bertoni plant andits use in various food products and beverages.

2. Description of the Related Art

Nowadays sugar alternatives are receiving increasing attention due toawareness of many diseases in conjunction with consumption of high-sugarfoods and beverages. However many artificial sweeteners such as dulcin,sodium cyclamate and saccharin were banned or restricted in somecountries due to concerns on their safety. Therefore non-caloricsweeteners of natural origin are becoming increasingly popular. Thesweet herb Stevia rebaudiana Bertoni, produces a number of diterpeneglycosides which feature high intensity sweetness and sensory propertiessuperior to those of many other high potency sweeteners.

The above-mentioned sweet glycosides, have a common aglycon, steviol,and differ by the number and type of carbohydrate residues at the C13and C19 positions. The leaves of Stevia are able to accumulate up to10-20% (on dry weight basis) steviol glycosides. The major glycosidesfound in Stevia leaves are Rebaudioside A (2-10%), Stevioside (2-10%),and Rebaudioside C (1-2%). Other glycosides such as Rebaudioside B, D,E, and F, Steviolbioside and Rubusoside are found at much lower levels(approx. 0-0.2%).

Two major glycosides—Stevioside and Rebaudioside A, were extensivelystudied and characterized in terms of their suitability as commercialhigh intensity sweeteners. Stability studies in carbonated beveragesconfirmed their heat and pH stability (Chang S. S., Cook, J. M. (1983)Stability studies of stevioside and Rebaudioside A in carbonatedbeverages. J. Agric. Food Chem. 31: 409-412.)

Steviol glycosides differ from each other not only by molecularstructure, but also by their taste properties. Usually stevioside isfound to be 110-270 times sweeter than sucrose, Rebaudioside A between150 and 320 times, and Rebaudioside C between 40-60 times sweeter thansucrose. Dulcoside A is 30 times sweeter than sucrose. Rebaudioside Ahas the least astringent, the least bitter, and the least persistentaftertaste thus possessing the most favorable sensory attributes inmajor steviol glycosides (Tanaka O. (1987) Improvement of taste ofnatural sweetners. Pure Appl. Chem. 69:675-683; Phillips K. C. (1989)Stevia: steps in developing a new sweetener. In: Grenby T. H. ed.Developments in sweeteners, vol. 3. Elsevier Applied Science, London.1-43.)

Methods for the extraction and purification of sweet glycosides from theStevia rebaudiana plant using water or organic solvents are describedin, for example, U.S. Pat. Nos. 4,361,697; 4,082,858; 4,892,938;5,972,120; 5,962,678; 7,838,044 and 7,862,845.

However, even in a highly purified state, steviol glycosides stillpossess undesirable taste attributes such as bitterness, sweetaftertaste, licorice flavor, etc. One of the main obstacles for thesuccessful commercialization of stevia sweeteners are these undesirabletaste attributes. It was shown that these flavor notes become moreprominent as the concentration of steviol glycosides increases (PrakashI., DuBois G. E., Clos J. F., Wilkens K. L., Fosdick L. E. (2008)Development of rebiana, a natural, non-caloric sweetener. Food Chem.Toxicol., 46, S75-S82.)

Some of these undesirable properties can be reduced or eliminated bysubjecting steviol glycosides to the reaction of intermoleculartransglycosylation, when new carbohydrate residues are attached toinitial molecule at C13 and C19 positions. Depending on the number ofcarbohydrate residues in these positions the quality and potency of thecompounds taste will vary.

Pullulanase, isomaltase (Lobov S. V., Jasai R., Ohtani K., Tanaka O.Yamasaki K. (1991) Enzymatic production of sweet stevioside derivatives:transglycosylation by glucosidases. Agric. Biol. Chem. 55: 2959-2965),β-galactosidase (Kitahata S., Ishikawa S., Miyata T., Tanaka O. (1989)Production of rubusoside derivatives by transglycosylation of variousβ-galactosidase. Agric. Biol. Chem. 53: 2923-2928), and dextransaccharase (Yamamoto K., Yoshikawa K., Okada S. (1994) Effectiveproduction of glucosyl-stevioside by α-1,6-transglucosylation of dextrandextranase. Biosci. Biotech. Biochem. 58: 1657-1661) have been used astransglycosylating enzymes, together with pullulan, maltose, lactose,and partially hydrolyzed starch, respectively, as donors of glycosidicresidues.

The transglucosylation of steviol glycosides was also performed byaction of cyclodextrin glucanotransferases (CGTase) produced by Bacillusstearothermophilus (U.S. Pat. Nos. 4,219,571, and 7,807,206) as a resultα-1,4-glucosyl derivatives were formed with degree of polymerization upto 10.

It was shown that the taste profile and sweetness power of glucosylderivatives are largely dependent on number of additional glucosylderivatives, i.e. the degree of polymerization of the α-1,4-glucosylchain. The increase in number of α-1,4-glucosyl residues improved thetaste quality but at the same time reduced the sweetness level. Thetreatment of transglucosylated stevioside with β-amylase resulted in aproduct consisting of mono- or di-α-1,4-glucosyl derivatives with bettertaste profile and optimal sweetness level (Tanaka, 1987). However insuch process, the resulting product contains high level of initialunreacted (unmodified) glycosides (generally >20%) which makes it notcompliant with regulatory requirements of less than 15% unreactedglycosides (α-Glucosyltransferase Treated Stevia, Japan's Specificationsand Standards for Food Additives, VIII edition, 2009, p. 257). Thereforeadditional steps for chromatographic separation of unreacted steviolglycosides are used to reduce initial unreacted (unmodified) glycosides'content. However chromatographic separation techniques generally involvehigh cost and are not suitable for large scale production.

It is to be noted also that many glucosyl stevia products contain up to20% residual dextrins which do not possess significant functionalproperties and reduce the content of steviol glycosides in the product.

Therefore it is necessary to develop simple process of preparation ofhigh purity glucosyl stevia products with optimal α-1,4-glucosyl chainlength and low unreacted glycosides level.

SUMMARY OF THE INVENTION

The present invention is aimed to overcome the disadvantages of existingStevia sweeteners. The invention describes a process for producing ahigh purity food ingredient from the extract of the Stevia rebaudianaBertoni plant and use thereof in various food products and beverages asa sweetness and flavor modifier.

The invention, in part, pertains to an ingredient comprisingglucosylated derivatives of steviol glycosides of Stevia rebaudianaBertoni plant. The steviol glycodsides are selected from the groupconsisting of stevioside, Rebaudioside A, Rebaudioside B, RebaudiosideC, Rebaudioside D, Rebaudioside E, Rebaudioside F, dulcoside A,steviolbioside, rubusoside, as well as other steviol glycosides found inStevia rebaudiana Bertoni plant and mixtures thereof.

The invention, in part, pertains to a process for producing aningredient containing glucosylated forms of stevioside, Rebaudioside A,Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E,Rebaudioside F, dulcoside A, steviolbioside, rubusoside, as well asother steviol glycosides found in Stevia rebaudiana Bertoni plant. Theprocess can be an enzymatic transglucosylating process using CGTasesproduced by cultures of Bacillus stearothermophilus. The process mayinclude the step of shortening glucosyl chains by β-amylase. The processcan also have the steps of decolorizing, desalting and removingmaltooligosaccharides and unmodified (unreacted) steviol glycosides. Thedecolorizing can be performed using activated carbon. The desalting canbe performed by passing through ion exchange resins and/or membranefilters. Removing the maltooligosaccharides can be performed by passingthrough macroporuos polymeric resin. Removing unmodified (unreacted)steviol glycosides can be performed by suspending the product in aqueousalcohol.

In the invention, Stevia extract commercialized by PureCircle (JiangXi)Co., Ltd. (China), containing stevioside (28-30%), Rebaudioside A(50-55%), Rebaudioside C (9-12%), Rebaudioside F (1-3%) and otherglycosides amounting to total steviol glycosides' content of at least95%, was used as a starting material. Alternatively stevia extracts withdifferent ratio of steviol glycosides as well as highly purified steviolglycosides such as Rebaudioside A, stevioside, Rebaudioside D,rubusoside etc, may be used as starting materials.

The starting material was subjected to the enzymatic transglucosylationby action of cyclodextrin glycosyltransferase (CGTase) in the presenceof starch as a glucose donor. As a result α-1,4-glucosyl derivativeswere formed with degree of polymerization up to 10. Then the formedderivatives were subjected to treatment with β-amylase to produceα-1,4-glucosyl derivatives possessing a degree of polymerization up to2.

The oligosaccharides from obtained reaction mixture were removed byAmberlite XAD7 HP resin, and then decolorized, deionized, concentratedand spray dried.

The unreacted steviol glycosides were subsequently removed by suspendingthe dried product in organic solvent and separating the suspended solidscontaining decreased level of unreacted steviol glycosides.

The obtained products were applied in various foods and beverages assweeteners, sweetener enhancers and flavor modifiers, including icecream, cookies, bread, fruit juices, milk products, baked goods andconfectionary products.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory and areintended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a furtherunderstanding of the invention. The drawings illustrate embodiments ofthe invention and together with the description serve to explain theprinciples of the embodiments of the invention.

FIG. 1 shows a high-performance liquid chromatographic chromatogram ofpurified transglucosylated Stevia extract without β-amylase treatmentcontaining long-chain α-1,4-glucosyl-derivatives with up to nineα-1,4-glucosyl residues;

FIG. 2 shows a high-performance liquid chromatographic (HPLC) chart ofβ-amylase treated product containing mono- anddi-α-1,4-glucosyl-derivatives of steviol glycosides, as well as highlevel of unreacted steviol glycoside;

FIG. 3 shows a high-performance liquid chromatographic (HPLC) chart ofβ-amylase treated product containing mono- anddi-α-1,4-glucosyl-derivatives of steviol glycosides, as well as lowlevel of unreacted steviol glycosides.

FIG. 4 shows a high-performance liquid chromatographic chromatogram oftransglucosylated Rebaudioside M containing α-1,4-glucosyl-derivativesof Rebaudioside M.

DETAILED DESCRIPTION OF THE INVENTION

Advantages of the present invention will become more apparent from thedetailed description given hereinafter. However, it should be understoodthat the detailed description and specific examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly, since various changes and modifications within the spirit andscope of the invention will become apparent to those skilled in the artfrom this detailed description.

Stevia extract commercialized by PureCircle (JiangXi) Co., Ltd. (China),containing stevioside (28-30%), Rebaudioside A (50-55%), Rebaudioside C(9-12%), Rebaudioside F (1-3%) and other glycosides (hereinaftercollectively, “steviol glycosides”) amounting to total steviolglycosides content of at least 95%, was used as a starting material.Alternatively stevia extracts with different ratio of steviol glycosidesas well as highly purified steviol glycosides such as Rebaudioside A,stevioside, Rebaudioside D, rubusoside etc, may be used as startingmaterials.

The HPLC analysis of the raw materials and products was performed onAgilent Technologies 1200 Series (USA) liquid chromatograph, equippedwith Zorbax-NH₂ (4.6×250 mm) column. The mobile phase wasacetonitrile-water gradient from 80:20, v/v (0-2 min) to 50:50, v/v(2-70 min). A diode array detector set at 210 nm was used as thedetector.

The transglucosylation was accomplished by cyclomaltodextringlucanotransferases (CGTases; EC 2.4.1.19) produced by Bacillusstearothermophilus St-100 (PureCircle Sdn Bhd Collection of IndustrialMicroorganisms—Malaysia). However, any other CGTase or enzyme possessingintermolecular transglucosylation activity may be applied as well. Theenzyme can be in a form of cell-free culture broth, concentrated liquidcell-free culture broth, spray dried or freeze dried cell-free culturebroth, or high purity protein. Free and immobilized enzyme preparationscan be used.

The activity of CGTase preparations was determined according to theprocedure described in Hale W. S., Rawlins L. C. (1951) Amylase ofBacillus macerans. Cereal Chem. 28, 49-58.

Starches of different origin may be used as donors of glucosyl unitssuch as, derived from wheat, corn, potato, tapioca, and sago.

Starch was subjected to partial hydrolysis (liquefaction) prior to thetransglycosylation reaction. The dextrose equivalent of the partiallyhydrolyzed starch can be in the range of about 10-25, preferably about12-16. Any enzyme capable of starch hydrolysis may be used forliquefaction, such as α-amylases, β-amylases etc. In one embodiment,CGTase and α-amylase mixtures as liquefying enzymes are preferred.

α-Amylase activity is expressed in Kilo Novo α-amylase Units (KNU). OneKNU is the amount of α-amylase which, under standard conditions (pH 7.1;37° C.), dextrinizes 5.26 g starch dry substance per hour.

The liquefaction mixture contains about 0.001-0.2 KNU, preferably about0.05-0.1 KNU of α-amylase per one unit of CGTase.

The use of α-amylase in liquefaction allows achieving higher throughputsin further activated carbon filtration. When the CGTase is used as theonly liquefying enzyme the filtration rate is approximately 10-15 L/hrper 1 m² of filter surface. In case of liquefaction enzyme mixture(comprising α-amylase and CGTase) the filtration rate is twice asfast—approximately 20-30 L/hr per 1 m² of filter surface.

The ratio of starch and CGTase in the liquefaction mixture is about0.1-0.5 units per one gram of starch, preferably about 0.2-0.4 units pergram.

The concentration of starch in liquefaction mixture is about 15-40%(wt/wt), preferably about 20-30%.

The liquefaction is conducted at about 70-90° C. during about 0.5-5hours, preferably about 1-2 hours.

After liquefaction, the reaction mixture is subjected to thermalinactivation of α-amylase at low pH conditions. The preferred pH rangefor inactivation is about pH 2.5 to pH 3.0 and preferred temperature isabout 95-105° C. The duration of thermal inactivation is about 5-10minutes.

After the inactivation, the pH of the reaction mixture is adjusted toabout pH 5.5-6.5 and the steviol glycosides are added to the mixture anddissolved. The preferred ratio of steviol glycosides to starch (kg ofsteviol glycosides per 1 kg of starch) is about 0.5-1.5, preferablyabout 0.8-1.2.

A second portion of CGTase preparation is added and thetransglucosylation reaction is conducted at about 65° C. for about 24-48hours. The amount of the second portion of CGTase is about 0.2-4 unitsof CGTase per gram of solids, preferably about 0.5-1.2 units per gram ofsolids.

Upon completion of transglucosylation reaction, about 30-50 units pergram of solids of β-amylase was added and the reaction was continued forabout 12-16 hours at about 35-55° C., preferably about 45° C. β-Amylaseproduced by Bacillus polymyxa St-3504 (PureCircle Sdn Bhd Collection ofIndustrial Microorganisms—Malaysia) was used in this stage. Howeverβ-amylases, and other starch hydrolyzing enzymes derived from any othersource including soybean, barley, bacterial, fungal and others may beused as well.

β-Amylase activity unit (1 AUN) is defined as the activity whichliberates 100 μg of reducing sugar (expressed by dextrose equivalent)per minute under the following conditions: 1 mL of enzyme solution ismixed with 5 mL of 1.2% starch solution (pH 5.5, M/20 Acetate Buffer)and kept for 20 min at 40° C.

The reaction was stopped by heating at about 95° C. for about 15 minutesto inactivate the enzymes, and the solution was treated with activatedcarbon, to obtain decolorized reaction mixture. The amount of activatedcarbon was about 0.02-0.4 grams per gram of solids, preferably about0.05-0.2 grams per gram of solids.

The decolorized reaction mixture was desalted by passing through ionexchange resins, such as Amberlite FPC23 (H⁺ type) and Amberlite FPA51(OH⁻ type). Other appropriate decolorizing and desalting methods, suchas membrane filtration, or other methods known in the art can be used.

The desalted reaction mixture was further concentrated by vacuumevaporator and dried by means of a spray dryer. Other appropriateconcentrating and drying methods, such as membrane filtration, freezedrying, or other methods known to art can be used.

The dried powder was suspended in aqueous alcohol. The powder to aqueousalcohol ratio (wt/vol) was 1:1 to 1:20, preferably 1:3 to 1:10. Theaqueous alcohol contained 0-50% (vol), preferably 1-10% water. Thesuspension is agitated at 30-100° C., preferably 50-85° C. during 1-24hours, preferably 2-15 hours. Then the suspended solids are separated bymeans of filtration. Any other technique known in the art suitable forseparating suspended solids from liquid such as centrifugation,decanting, etc. can be used. The obtained solids are dried in rotarydrum vacuum drier. Any other dryer known t in the art may be used aswell. Alternatively the separated solids may be dissolved in water,evaporated from traces of alcohol and spray dried.

The alcohols employed in the invention may be selected from the groupconsisting of alkanols, and are preferably selected from the groupincluding methanol, ethanol, n-propanol, 2-propanol, 1-butanol, and2-butanol.

The resulting product contains low level non-modified glycosides,short-chain (containing two or less α-1,4-glucosyl residues) derivativesand a mixture of maltooligosaccharides (Sample 1). As used herein, theexpressions “low level non-modified glycosides” or “low level unreactedglycosides” shall refer to glycoside levels of less than about 20%, andpreferably less than about 15%, on an anhydrous basis. In someembodiments, an unreacted glycoside level of about 12%, about 10% oreven lower can be attained using this method.

In order to prepare a product with higher content of total sweetglycosides (the sum of glycosylated and non-glycosylated glycosides),the maltooligosaccharides were removed using Amberlite XAD7 HP prior tothe desalting treatment. The steviol glycosides and their glucosylatedderivatives were adsorbed on the resin and subsequently eluted byaqueous ethanol. The resulted aqueous ethanol eluate, containingglucosyl steviol glycosides, was subsequently decolorized and desaltedas described above and the glycosides solution, after the evaporation ofeluting solvent, was powdered by spray drying. The dried powder wassuspended in aqueous alcohol and processed as described above to removeunmodified (unreacted) steviol glycosides. The resulting productcontains low level non-modified glycosides, and short-chain (containingtwo or less α-1,4-glucosyl residues) derivatives (Sample 2).

The embodiments of the invention exemplified by Samples 1 and 2 are freeor substantially free of higher glucosylated derivatives having morethan 2 glucosyl residues. In accordance with this invention, the highlypurified glucosyl stevia composition preferably comprises greater thanabout 50% by weight mono-, and diglucosyl steviol glycosides.

Using a similar process as for Sample 2, with exclusion of the β-amylaseand aqueous alcohol treatment stages, a product containing non-modifiedglycosides and long chain α-1,4-glucosyl-derivatives (with up to nineα-1,4-glucosyl residues) was prepared (Sample 3).

Using a similar process as for sample 2, with exclusion of the aqueousalcohol treatment stage, a product containing high level non-modifiedglycosides, and short-chain derivatives (containing two or lessα-1,4-glucosyl residues) was prepared (Sample 4). The composition of thesamples is summarized in Table 1.

TABLE 1 Composition of glucosyl steviol glycosides samples Content, %Compounds Sample 1 Sample 2 Sample 3 Sample 4 Stevioside 2.4 3.2 3.113.2 Rebaudioside C 0.7 1.0 1.0 3.0 Rebaudioside A 5.6 7.5 6.1 12.3Monoglucosyl-stevioside 16.2 21.9 7.5 22.2 (StevG1) Monoglucosyl- 20.928.1 11.2 22.4 Rebaudioside A (RebAG1) Diglucosyl-stevioside 10.1 13.68.5 8.9 (StevG2) Diglucosyl-Rebaudioside 13.8 18.6 9.7 11.4 A (RebAG2)Higher glucosylated 1.3 1.7 48.8 1.8 derivatives Total content of 8.711.7 10.2 28.5 unreacted glycosides Total content of 71.0 95.5 95.8 95.3glycosides

The sensory assessment of samples was carried using aqueous solutions,with 20 panelists. Based on overall acceptance the most desirable andmost undesirable samples were chosen. The results are shown in Table 2.

TABLE 2 Sensory assessment of samples in water system Judgment Sample 1Sample 2 Sample 3 Sample 4 Most desirable 6 10 1 3 Most undesirable 1 012 7 Sweetness power 150 160 110 160 Comments Sweet, light, Sweet,light, Sweet, Sweet, soft, round, soft, round, slightly slightlypleasant, pleasant, bitter, bitter, almost similar to astringent,astringent, similar to sucrose, no slight slight sucrose, no lingeringlingering lingering lingering aftertaste, aftertaste, aftertaste,aftertaste, sweetness sweetness sweetness sweetness onset is onset isonset is slow onset is rapid moderate rapid

As apparent from the results in Table 2, the sweetness quality of theSamples 1 and 2 was rated as most superior. Overall the samples withshort-chain (containing two or less α-1,4-glucosyl residues) derivativesand low level of unreacted glycosides (Samples 1 and 2) possessed bettertaste profiles compared to samples with long-chain glucosyl derivatives(Sample 3) and short-chain (containing two or less α-1,4-glucosylresidues) derivatives and high level of unreacted glycosides (Sample 4).

Samples 1 and 2 show comparable sweetness power (150-160 times sweetercompared to a 5% sucrose solution) with control Sample 4 (160 times);however their flavor profile was clearly superior to the control Sample4.

The compositions can be used as sweetness enhancers, flavor enhancersand sweeteners in various food and beverage products. Non-limitingexamples of food and beverage products include carbonated soft drinks,ready to drink beverages, energy drinks, isotonic drinks, low-caloriedrinks, zero-calorie drinks, sports drinks, teas, fruit and vegetablejuices, juice drinks, dairy drinks, yoghurt drinks, alcohol beverages,powdered beverages, bakery products, cookies, biscuits, baking mixes,cereals, confectioneries, candies, toffees, chewing gum, dairy products,flavored milk, yoghurts, flavored yoghurts, cultured milk, soy sauce andother soy base products, salad dressings, mayonnaise, vinegar,frozen-desserts, meat products, fish-meat products, bottled and cannedfoods, tabletop sweeteners, fruits and vegetables.

Additionally the compositions can be used in drug or pharmaceuticalpreparations and cosmetics, including but not limited to toothpaste,mouthwash, cough syrup, chewable tablets, lozenges, vitaminpreparations, and the like.

The compositions can be used “as-is” or in combination with othersweeteners, flavors and food ingredients.

Non-limiting examples of sweeteners include steviol glycosides,stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, RebaudiosideD, Rebaudioside E, Rebaudioside F, dulcoside A, steviolbioside,rubusoside, as well as other steviol glycosides found in Steviarebaudiana Bertoni plant and mixtures thereof, stevia extract, Luo HanGuo extract, mogrosides, high-fructose corn syrup, corn syrup, invertsugar, fructooligosaccharides, inulin, inulooligosaccharides, couplingsugar, maltooligosaccharides, maltodextins, corn syrup solids, glucose,maltose, sucrose, lactose, aspartame, saccharin, sucralose, sugaralcohols.

Non-limiting examples of flavors include lemon, orange, fruity, banana,grape, pear, pineapple, bitter almond, cola, cinnamon, sugar, cottoncandy, vanilla flavors.

Non-limiting examples of other food ingredients include flavors,acidulants, organic and amino acids, coloring agents, bulking agents,modified starches, gums, texturizers, preservatives, antioxidants,emulsifiers, stabilisers, thickeners, gelling agents.

The following examples illustrate various embodiments of the invention.It will be understood that the invention is not limited to thematerials, proportions, conditions and procedures set forth in theexamples, which are only illustrative.

Example 1 Preparation of CGTase

A strain of Bacillus stearothermophilus St-100 was inoculated in 2,000liters of sterilized culture medium containing 1.0% starch, 0.25% cornextract, 0.5% (NH₄)₂SO₄, and 0.2% CaCO₃ (pH 7.0-7.5) at 56° C. for 24hrs with continuous aeration (2,000 L/min) and agitation (150 rpm). Theobtained culture broth was filtered using Kerasep 0.1 μm ceramicmembrane (Novasep, France) to separate the cells. The cell-free permeatewas further concentrated 2-fold on Persep 10 kDa ultrafilters (Orelis,France). The activity of the enzyme was determined according to Hale,Rawlins (1951). A crude enzyme preparation with activity of about 2unit/mL was obtained.

Example 2 Preparation of β-Amylase

A strain of Bacillus polymyxa St-3504 was inoculated in 2,000 liters ofsterilized culture medium containing 1.0% starch, 0.5% peptone, 0.5%corn extract, 0.5% NaCl, 0.02% MnSO₄ and 0.1% CaCO₃ (pH 7.0-7.5) at 32°C. for 24 hrs with continuous aeration (2,000 L/min) and agitation (150rpm). The obtained culture broth was filtered using Kerasep 0.1 μmceramic membrane (Novasep, France) to separate the cells. 10% of glucosewas added to the cell-free permeate which was further concentrated onPersep 10 kDa ultrafilters (Orelis, France) and dried using Alpha 1-4LSC freeze drier unit (Christ, Germany) to obtain a powder with 20,000AUN/g activity. β-Amylase activity unit (1 AUN) was defined as theactivity which liberates 100 mg of reducing sugar (expressed by dextroseequivalent) per minute under the following conditions: 1 mL of enzymesolution is mixed with 5 mL of 1.2% starch solution (pH 5.5, M/20Acetate Buffer) and kept for 20 min at 40° C.

Example 3 Preparation of Short-Chain Glucosyl Stevia Composition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to EXAMPLE 1 were added, and the liquefactionof starch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extractproduced by PureCircle (JiangXi) Co., Ltd. (China), containingstevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%,Rebaudioside F (1.7%) and other glycosides amounting to total steviolglycosides content of about 96.4% was added to liquefied starch andstirred until a homogeneous solution was obtained. 200 units of CGTasewas added to the solution and the mixture was held at a temperature of65° C. for 24 hours under continuous agitation. Then the temperature wasreduced to 45° C., and 8,000 units of β-amylase obtained according toEXAMPLE 2 was added to reaction mixture. The reaction was continued foranother 12 hours. The obtained reaction mixture was heated at 95° C. for15 minutes to inactivate the enzymes. 20 grams of activated carbon wasadded and the mixture was heated to 75° C. and held for 30 minutes. Themixture was filtered and the filtrate was diluted with water to 5%solids content and passed through columns packed with Amberlite FPC23(H⁺) and Amberlite FPA51 (OH⁻) ion exchange resins. The desaltedsolution was concentrated at 60° C. under vacuum, and dried into apowder form using laboratory spray dryer. The dried powder was suspendedin 5 volumes of 95% aqueous ethanol. The suspension was agitated at 80°C., during 12 hours. Then the suspended solids were separated byfiltration. The obtained solids were dried in vacuum dryer at 90° C.during 5 hours. 170 grams of product was obtained (Sample 1).

Example 4 Preparation of Highly Purified Short-Chain Glucosyl SteviaComposition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to EXAMPLE 1 were added, and the liquefactionof starch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extractproduced by PureCircle (JiangXi) Co., Ltd. (China), containingstevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%,Rebaudioside F (1.7%) and other glycosides amounting to total steviolglycosides content of about 96.4% was added to liquefied starch andstirred until a homogeneous solution was obtained. 200 units of CGTasewas added to the solution and the mixture was held at a temperature of65° C. for 24 hours under continuous agitation. Then the temperature wasreduced to 45° C., and 8,000 units of β-amylase obtained according toEXAMPLE 2 was added to reaction mixture. The reaction was continued foranother 12 hours. The obtained reaction mixture was heated at 95° C. for15 minutes to inactivate the enzymes. 20 grams of activated carbon wasadded and the mixture was heated to 75° C. and held for 30 minutes. Themixture was filtered and the filtrate was diluted with water to 5%solids content and passed through columns each packed with 4000 mLAmberlite XAD 7HP macroporous adsorbent resin. The columns were washedwith 5 volumes of water and 2 volumes of 20% (v/v) ethanol. The adsorbedglycosides were eluted with 50% ethanol. Obtained eluate was passedthrough columns packed with Amberlite FPC23 (H⁺) and Amberlite FPA51(OH⁻) ion exchange resins. The ethanol was evaporated and the desaltedand decolorized water solution was concentrated at 60° C. under vacuum,then dried into a powder form using laboratory spray dryer. The driedpowder was suspended in 5 volumes of 95% aqueous ethanol. The suspensionwas agitated at 80° C., during 12 hours. Then the suspended solids wereseparated by filtration. The obtained solids were dried in vacuum dryerat 90° C. during 5 hours. 121 grams of product was obtained (Sample 2).

Example 5 Preparation of Highly Purified Long-Chain Glucosyl SteviaComposition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to EXAMPLE 1 were added, and the liquefactionof starch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extractproduced by PureCircle (JiangXi) Co., Ltd. (China), containingstevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%,Rebaudioside F (1.7%) and other glycosides amounting to total steviolglycosides content of about 96.4% was added to liquefied starch andstirred until a homogeneous solution was obtained. 200 units of CGTasewas added to the solution and the mixture was held at a temperature of65° C. for 24 hours under continuous agitation. The obtained reactionmixture was heated at 95° C. for 15 minutes to inactivate the enzyme. 20grams of activated carbon was added and the mixture was heated to 75° C.and held during 30 min. The mixture was filtered and the filtrate wasdiluted with water to 5% solids content and passed through columns eachpacked with 4000 mL Amberlite XAD 7HP macroporous adsorbent resin. Thecolumns were washed with 5 volumes of water and 2 volumes of 20% (v/v)ethanol. The adsorbed glycosides were eluted with 50% ethanol. Obtainedeluate was passed through columns packed with Amberlite FPC23 (H⁺) andAmberlite FPA51 (OH⁻) ion exchange resins. The ethanol was evaporatedand the desalted and decolorized water solution was concentrated at 60°C. under vacuum, then dried into a powder form using laboratory spraydryer. 165 grams of product was obtained (Sample 3).

Example 6 Preparation of Highly Purified Short-Chain Glucosyl SteviaComposition

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to EXAMPLE 1 were added, and the liquefactionof starch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extractproduced by PureCircle (JiangXi) Co., Ltd. (China), containingstevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%,Rebaudioside F (1.7%) and other glycosides amounting to total steviolglycosides content of about 96.4% was added to liquefied starch andstirred until a homogeneous solution was obtained. 200 units of CGTasewas added to the solution and the mixture was held at a temperature of65° C. for 24 hours under continuous agitation. Then the temperature wasreduced to 45° C., and 8,000 units of β-amylase obtained according toEXAMPLE 2 was added to reaction mixture. The reaction was continued foranother 12 hours. The obtained reaction mixture was heated at 95° C. for15 minutes to inactivate the enzymes. 20 grams of activated carbon wasadded and the mixture was heated to 75° C. and held for 30 minutes. Themixture was filtered and the filtrate was diluted with water to 5%solids content and passed through columns each packed with 4000 mLAmberlite XAD 7HP macroporous adsorbent resin. The columns were washedwith 5 volumes of water and 2 volumes of 20% (v/v) ethanol. The adsorbedglycosides were eluted with 50% ethanol. Obtained eluate was passedthrough columns packed with Amberlite FPC23 (H⁺) and Amberlite FPA51(OH⁻) ion exchange resins. The ethanol was evaporated and the desaltedand decolorized water solution was concentrated at 60° C. under vacuum,then dried into a powder form using laboratory spray dryer. 154 grams ofproduct was obtained (Sample 4).

Example 7 Low-Calorie Orange Juice Drink

Orange concentrate (35%), citric acid (0.35%), ascorbic acid (0.05%),orange red color (0.01%), orange flavor (0.20%), Rebaudioside A (0.003%)and different glucosyl stevia compositions (0.03%) were blended anddissolved completely in water (up to 100%) and pasteurized. Glucosylstevia compositions were represented by Samples 1, 2, 3 and 4, obtainedaccording to EXAMPLES 3, 4, 5 and 6, respectively.

The sensory evaluations of the samples are summarized in Table 3. Thedata show that the best results can be obtained by using the high purityshort-chain glucosyl stevia compositions (containing two or lessα-1,4-glucosyl residues and low unreacted steviol glycosides) (Samples 1and 2). Particularly the drinks prepared with Samples 1 and 2 exhibiteda rounded and complete flavor profile and mouthfeel.

TABLE 3 Evaluation of orange juice drink samples Comments Sample FlavorAftertaste Mouthfeel No. 1 High quality sweetness, Clean, almost no Fullpleasant taste similar to bitterness, no aftertaste sucrose, rounded andbalanced flavor No. 2 High quality sweetness, Clean, no bitterness andFull pleasant taste similar to no aftertaste sucrose, rounded andbalanced flavor No. 3 High quality sweetness, Clean, almost no Almostpleasant taste almost bitterness, no aftertaste acceptable similar tosucrose, rounded and balanced flavor No. 4 Sweet, licorice notes Slightbitterness and Not aftertaste acceptable

The same method can be used to prepare juices and juice drinks fromother fruits, such as apples, lemons, apricots, cherries, pineapples,mangoes, etc.

Example 8 Low-Calorie Carbonated Beverage

A carbonated beverage according to formula presented below was prepared.

Ingredients Quantity, % Sucrose 5.5 Cola flavor 0.340 ortho-Phosphoricacid 0.100 Sodium citrate 0.310 Sodium benzoate 0.018 Citric acid 0.018Rebaudioside A 0.003 Glucosyl stevia composition 0.05 Carbonated waterto 100

The sensory properties were evaluated by 20 panelists. The results aresummarized in Table 4.

TABLE 4 Evaluation of low-calorie carbonated beverage samples Number ofpanelists detected the attribute Sample Sample Sample Sample No. 1 No. 2No. 3 No. 4 Taste attribute Bitter taste 0 0 10 12 Astringent 1 0 15 15taste Aftertaste 1 0 13 18 Comments Quality of Clean Clean Clean Bitteraftertaste sweet taste (18 of 20) (20 of 20) (14 of 20) (10 of 20)Overall Satisfactory Satisfactory Satisfactory Satisfactory evaluation(19 of 20) (20 of 20) (11 of 20) (9 of 20)

The above results show that the beverages prepared using Samples 1 and 2possessed the best organoleptic characteristics.

Example 9 Diet Cookies

Flour (50.0%), margarine (30.0%) fructose (10.0%), maltitol (8.0%),whole milk (1.0%), salt (0.2%), baking powder (0.15%), vanillin (0.1%)and different glucosyl stevia compositions (0.03%) were kneaded well indough-mixing machine. The obtained dough was molded and baked in oven at200° C. for 15 minutes. Glucosyl stevia compositions were represented bySamples 1, 2, 3 and 4, obtained according to EXAMPLES 3, 4, 5 and 6,respectively.

The sensory properties were evaluated by 20 panelists. The best resultswere obtained in samples prepared by high purity short-chain glucosylstevia compositions (containing two or less α-1,4-glucosyl residues)derivatives (Samples 1 and 2). The panelists noted rounded and completeflavor profile and mouthfeel in cookies prepared with Samples 1 and 2.

Example 10 Yoghurt

Different glucosyl stevia compositions (0.03%) and sucrose (4%) weredissolved in low fat milk. Glucosyl stevia compositions were representedby Samples 1, 2, 3 and 4, obtained according to EXAMPLES 3, 4, 5 and 6,respectively. After pasteurizing at 82° C. for 20 minutes, the milk wascooled to 37° C. A starter culture (3%) was added and the mixture wasincubated at 37° C. for 6 hours then at 5° C. for 12 hours.

The sensory properties were evaluated by 20 panelists. The best resultswere obtained in samples prepared by high purity short-chain glucosylstevia compositions (containing two or less α-1,4-glucosyl residues)derivatives (Samples 1 and 2). The panelists noted rounded and completeflavor profile and mouthfeel in samples prepared with Samples 1 and 2.

As noted above, the invention is directed to glucosylated derivatives ofsteviol glycosides of the Stevia rebaudiana plant, including any steviolglycoside found in S. rebaudiana such as Rebaudioside M, (also known asRebaudioside X). The Rebaudioside M (Reb M) α-1,4-glucosylatedderivatives (glucosyl Reb M) can have any number of α-1,4-glucosylresidues, including 1 to 10 α-1,4-glucosyl residues, or any combinationof derivatives with different number or position of α-1,4-glucosylresidues. For example, 40% of the Reb M glucosylated derivatives in aparticular product may have 4 or fewer α-1,4-glucosyl residues, 20% ofthe Reb M glucosylated derivatives in that product may have 6 to 10α-1,4-glucosyl residues, etc. The amount of total steviol glycosides(TSG) is a sum of amounts of glucosylated steviol glycosides (includingα-1,4-glucosyl Reb M) and unmodified steviol glycosides (includingunmodified Reb M). The weight ratio of the Reb M α-1,4-glucosylderivatives to the TSG in a product can have different values, forexample ranging from 1:1 to 1:10,000; such as 1:2; 1:10; 1:100; 1:500;1:900; 1:1,000; 1:5,000 etc.

Example 11 Preparation of Glucosyl Reb M

100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNUof α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units ofCGTase obtained according to EXAMPLE 1 were added, and the liquefactionof starch was carried out at 80° C. for about one hour to dextroseequivalent about 15. The pH of reaction mixture was adjusted to pH 2.8by hydrochloric acid and the mixture was boiled at 100° C. during 5minutes to inactivate the enzymes. After cooling to 65° C., the pH wasadjusted to pH 6.0 with sodium hydroxide solution. 100 g Rebaudioside M(also known as Rebaudioside X) produced by PureCircle Sdn. Bhd.(Malaysia), containing Rebaudioside M 83.68%, Rebaudioside D 9.45%,Rebaudioside A 2.12%, Rebaudioside B (1.53%), and other glycosidesamounting to total steviol glycosides content of about 96.85% wasdissolved in 9,000 mL water (pH was adjusted to pH 6.0 with NaOHsolution) and was added to liquefied starch and stirred until ahomogeneous solution was obtained. 200 units of CGTase was added to thesolution and the mixture was held at a temperature of 65° C. for 24hours under continuous agitation. The obtained reaction mixture washeated at 95° C. for 15 minutes to inactivate the enzyme. 20 grams ofactivated carbon was added and the mixture was heated to 75° C. and heldduring 30 min. The mixture was filtered and the filtrate was passedthrough columns each packed with 4000 mL Amberlite XAD 7HP macroporousadsorbent resin. The columns were washed with 5 volumes of water and 2volumes of 20% (v/v) ethanol. The adsorbed glycosides were eluted with50% ethanol. Obtained eluate was passed through columns packed withAmberlite FPC23 (H⁺) and Amberlite FPA51 (OH⁻) ion exchange resins. Theethanol was evaporated and the desalted and decolorized water solutionwas concentrated at 60° C. under vacuum, then dried into a powder formusing laboratory spray dryer. 159 grams of product was obtained (Sample5). The HPLC chromatogram of Sample 5 is provided in FIG. 4 and theassay results in Table 5.

TABLE 5 HPLC assay of Sample 5 Content, area % Compounds Sample 5Rebaudioside M 16.2 Rebaudioside D 3.6 Monoglucosyl-Rebaudioside M(RebMG1) 21.8 Monoglucosyl-Rebaudioside D (RebDG1) 3.5Diglucosyl-Rebaudioside M (RebMG2) 12.6 Higher glucosylated derivatives42.3 Total glucosylated derivatives of Rebaudioside M 76.7

Example 12 Evaluation of Glucosyl Reb M

The sensory assessment of Sample 5 prepared according top EXAMPLE 11,was carried using aqueous solutions, with 20 panelists. Sample 5 wasassessed along with glucosyl Rebaudioside A and glucosyl Steviosidesamples (Table 6 and 7) produced by PureCircle Sdn Bhd (Malaysia). Basedon overall acceptance the most desirable and most undesirable sampleswere chosen. The results are shown in Table 8.

TABLE 6 HPLC assay of Glucosyl Stevioside Content, area % GlucosylCompounds Stevioside Stevioside 14.3 Monoglucosyl-Stevioside (StevG1)21.8 Diglucosyl-Stevioside (StevG2) 20.1 Higher glucosylated derivatives43.8 Total glucosylated derivatives of stevioside 85.7

TABLE 7 HPLC assay of Glucosyl Reb A Content, area % Compounds GlucosylReb A Rebaudioside A 16.1 Monoglucosyl-Rebaudioside A (RebMG1) 20.6Diglucosyl-Rebaudioside A (RebMG2) 19.6 Higher glucosylated derivatives43.7 Total glucosylated derivatives of Rebaudioside A 83.9

TABLE 8 Sensory assessment of samples in water system Glucosyl Sample 5Judgment Stevioside Glucosyl Reb A (Glucosyl RebM) Most desirable 1 4 15Most undesirable 15 5 0 Sweetness power 120 160 160 Comments Sweet,Sweet, Sweet, light, bitter, slightly soft, round, astringent, bitter,pleasant, lingering astringent, similar to aftertaste, slight lingeringsucrose, no sweetness onset aftertaste, lingering is slow sweetnessonset aftertaste, is slow sweetness onset is rapid

As apparent from the results in Table 8, the sweetness quality of theSample 5 was rated as most superior for their flavor profile.

Example 13 Low-Calorie Orange Juice Drink

Orange concentrate (35%), citric acid (0.35%), ascorbic acid (0.05%),orange red color (0.01%), orange flavor (0.20%), Rebaudioside A (0.003%)and different glucosyl stevia compositions (0.03%) were blended anddissolved completely in water (up to 100%) and pasteurized. Glucosylstevia compositions were represented by glucosyl Rebaudioside A,glucosyl Stevioside and Sample 5, obtained according to EXAMPLE 11.

The sensory evaluations of the samples are summarized in Table 9. Thedata show that the best results can be obtained by using the Sample 5.Particularly the drink prepared with Sample 5 exhibited a rounded andcomplete flavor profile and mouthfeel.

TABLE 9 Evaluation of orange juice drink samples Comments Sample FlavorAftertaste Mouthfeel Glucosyl Sweet, licorice notes Bitterness and NotStevioside aftertaste acceptable Glucosyl Pleasant taste close to Slightbitterness and Not Reb A sucrose, off-notes aftertaste acceptable Sample5 High quality sweetness, Clean, no bitterness Full (Glucosyl pleasanttaste similar to and no aftertaste Reb M) sucrose, rounded and balancedflavor

The same method can be used to prepare juices and juice drinks fromother fruits, such as apples, lemons, apricots, cherries, pineapples,mangoes, etc.

It is to be understood that the foregoing descriptions and specificembodiments shown herein are merely illustrative of the best mode of theinvention and the principles thereof, and that modifications andadditions may be easily made by those skilled in the art withoutdeparting for the spirit and scope of the invention, which is thereforeunderstood to be limited only by the scope of the appended claims.

1. A composition comprising Rebaudioside M glucosyl derivatives havingat least one α-1,4-glucosyl residue.
 2. A composition of claim 1 whereinthe ratio of total steviol glycosides, comprising α-1,4-glucosylderivatives of steviol glycosides and unmodified steviol glycosides, toglucosyl derivatives of Rebaudioside M is in the range of 10,000:1 to1:1.
 3. A sweetener composition comprising composition of claim 1 and atleast one additional substance selected from the group consisting ofsteviol glycosides, stevioside, rebaudioside A, rebaudioside B,rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F,rebaudioside M, rebaudioside N, rebaudioside O, dulcoside A,steviolbioside, rubusoside, glycosylated steviol glycosides,glycosylated steviol glycosides as well as other steviol glycosidesfound in Stevia rebaudiana plant and mixtures thereof, stevia extract,Luo Han Guo extract, mogrosides, high-fructose corn syrup, corn syrup,invert sugar, fructooligosaccharides, inulin, inulooligosaccharides,coupling sugar, maltooligosaccharides, maltodextins, corn syrup solids,glucose, maltose, sucrose, lactose, aspartame, saccharin, sucralose,sugar alcohols and combination thereof.
 4. A flavor compositioncomprising composition of claim 1 and at least one additional flavoringagent selected from the group consisting of lemon, berry, orange, fruit,banana, grape, pear, pineapple, mango, bitter almond, cola, cinnamon,sugar, cotton candy, vanilla, terpenoid glycosides and combinationthereof.
 5. A food ingredient comprising composition of claim 1 and atleast one additional food ingredient selected from the group consistingof acidulants, organic and amino acids, coloring agents, bulking agents,modified starches, gums, texturizers, preservatives, antioxidants,emulsifiers, stabilisers, thickeners, gelling agents, and combinationthereof.
 6. A food, beverage, cosmetic, pharmaceutical product or otherconsumable comprising composition of claim 1.